This invention relates to AGP-antibody fusion proteins, DNA, and uses thereof.
ELAM-1 is an integral membrane adhesion protein. It possesses an extracellular domain including an N-terminal lectin-related segment, an epidermal growth factor related repeat, and multiple complement regulatory protein motifs (Bevilacqua et al., Science 243:1160, 1989; Stoolman et al., Cell 56:907, 1989). ELAM-1 is specifically expressed on the surface of endothelial cells activated by the cytokines IL1 and tumor necrosis factor (TNF) (Bevilacqua et al., Proc. Natl. Acad. Sci. USA 84:9238, 1987), or the peptide hormone Substance P (Matis et al., J. Invest. Dermatol. 94:492, 1990). It mediates adhesion of myeloid cells (e.g., neutrophilic granulocytes) to cytokine-activated endothelial cells (Bevilacqua et al., Proc. Natl. Acad. Sci USA 84:9238, 1987). It has been suggested that ELAM-1 is involved in the regulation of inflammatory and immunological events at the interface of the blood and the blood vessel wall (Bevilacqua et al., Science 243:1160, 1989).
In a first aspect, the invention features a method of inhibiting the binding of a cell bearing a cell adhesion protein to a molecule or cell bearing a carbohydrate determinant specific for the cell adhesion molecule. The method involves contacting the cell adhesion protein-bearing cell with an xcex11-acid glycoprotein (AGP)-antibody fusion protein bearing the carbohydrate determinant.
In a second aspect, the invention features a method of reducing inflammation in a human patient involving administering to the patient a therapeutically-effective amount of an AGP-antibody fusion protein bearing a sialyl-LeX determinant.
In a third aspect, the invention features an AGP-antibody fusion protein to which there is covalently bonded a carbohydrate determinant specific for a cell adhesion protein.
In related aspects, the invention features purified nucleic acid encoding an AGP-antibody fusion protein containing sites for the attachment of a carbohydrate determinant which is specific for a cell adhesion protein; a vector including such nucleic acid; and a recombinant cell including such a vector.
In preferred embodiments of each of the above aspects, the cell adhesion protein is a selectin, such as ELAM-1; the carbohydrate determinant is sialyl-LeX; the sialyl-LeX determinant is N-linked or O-linked; the AGP-antibody fusion protein contains multiple sialyl-LeX determinants; the AGP-antibody fusion protein includes, as an antibody portion, an IgG1 CH2, CH3, or hinge domain; the AGP-antibody fusion protein includes one or more of the N-linked glycan addition sites of xcex11-acid glycoprotein; the antibody portion of the AGP-antibody fusion protein bears one or more non-naturally occurring carbohydrate determinants; the carbohydrate determinant interferes with the antibody""s ability to fix complement and bind an Fc receptor (for example, due to a carbohydrate determinant attached to one or more of amino acids 274, 287, or 322 of the sequence shown in FIG. 1); and the AGP-antibody fusion protein is soluble.
In a final aspect, the invention features a method of protecting a mammal against any adverse immune reaction (including, without limitation, sepsis, organ damage attributable to inappropriate leukocyte extravasation, adult respiratory distress syndrome, glomerular nephritis, and rhumatoid arthritis), the method including administering to the mammal a therapeutically-effective amount of an AGP-antibody fusion protein. Preferably, the method involves treating a mammal for an adverse immune reaction which is induced by a microbial factor. Such microbial factors include, without limitation, gram-negative bacteria lipopolysaccharides (LPS), peptidoglycans from gram-positive organisms, mannan from fungal cell walls, polysaccharides, extracellular enzymes (e.g., streptokinase) and toxins (e.g., toxic shock enterotoxins of staphylococci). In other preferred embodiments, the method involves treating a mammal for any adverse immune reaction (see supra) which is induced by a host factor. Such host factors include, without limitation, metabolites of complement, kinin, and coagulation systems, factors released from stimulated cells (e.g., cytokines such as interleukin 1 (IL1) and tumor necrosis factor-xcex1 (TNF)), enzymes and oxidants from polymorphonuclear leukocytes (PMNs), vasopeptides (e.g., histamine), and products of the metabolism of arachidonic acid. In other preferred embodiments, the adverse immune reaction is induced by recombinant TNF-xcex1 or is induced by recombinant IL1. In yet other preferred embodiments, the adverse immune reaction is septic shock or is septicemia.
In other preferred embodiments, the AGP-antibody fusion protein bears one or more carbohydrate determinants capable of interfering with the antibody""s ability to fix complement and bind an Fc receptor; the antibody portion of the AGP-antibody fusion protein consists of the CH2 and CH3 antibody domains; and the antibody portion of the AGP-antibody fusion protein is an IgG1 domain.
By xe2x80x9ccell adhesion proteinxe2x80x9d is meant a protein, present at some point in its in vivo existence on the cell surface, which mediates a specific interaction with a protein (e.g., a protein bearing a carbohydrate ligand) on the surface of a second cell.
By xe2x80x9ccarbohydrate determinantxe2x80x9d is meant a moiety containing one or more carbohydrate groups which is present on a cell surface (at some point in its in vivo existence) and which interacts in a specific manner with a protein, e.g., a cell adhesion protein, e.g., on the surface of a second cell.
By xe2x80x9cselectinxe2x80x9d is meant a member of a family of cellular adhesion molecules that are characterized structurally by the presence of a lectin-like domain, an epidermal growth factor-like domain, a series of cysteine-rich repeats, a transmembrane domain and a short cytoplasmic tail.
By xe2x80x9cnon-naturally occurringxe2x80x9d is meant, in this case, any carbohydrate determinant that is not one which is naturally bound to the fusion protein at that amino acid location.
By xe2x80x9cinflammationxe2x80x9d is meant a pathologic process consisting of cytologic and histologic reactions that occur in the affected blood vessels and adjacent tissues in response to an injury or abnormal stimulation caused by a physical, chemical, or biologic agent.
By xe2x80x9cpurified nucleic acidxe2x80x9d is meant DNA that is free of the genes which, in the naturally-occurring genome of the organism from which the DNA of the invention is derived, flank the gene. The term therefore includes, for example, a recombinant DNA which is incorporated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryote or eukaryote; or which exists as a separate molecule (e.g., a cDNA or a genomic or cDNA fragment produced by PCR or restriction endonuclease digestion) independent of other sequences. It also includes a recombinant DNA which is part of a hybrid gene encoding additional polypeptide sequence.
By xe2x80x9cN-linkedxe2x80x9d is meant bonded to the amide nitrogen of an asparagine residue of a protein.
By xe2x80x9cO-linkedxe2x80x9d is meant bonded to the hydroxyl-group oxygen of a serine, threonine, or hydroxylysine residue of a protein.
The drawings will first briefly be described.